U.S. patent number 4,689,295 [Application Number 06/529,031] was granted by the patent office on 1987-08-25 for test for salmonella.
This patent grant is currently assigned to Integrated Genetics, Inc.. Invention is credited to Renee A. Fitts, Robert L. Taber.
United States Patent |
4,689,295 |
Taber , et al. |
August 25, 1987 |
**Please see images for:
( Certificate of Correction ) ** |
Test for Salmonella
Abstract
Method of detecting the presence of Salmonella in a food sample
including providing at least one DNA probe which is capable of
selectively hybridizing to Salmonella DNA to form detectable
complexes, contacting the DNA probes with the bacteria in the food
sample under conditions which allow the probe to hybridize to
Salmonella DNA present in the food sample to form hybrid DNA
complexes, and detecting the hybrid DNA complexes as an indication
of the presence of Salmonella in the food sample.
Inventors: |
Taber; Robert L. (Wellesley,
MA), Fitts; Renee A. (Framingham, MA) |
Assignee: |
Integrated Genetics, Inc.
(Framingham, MA)
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Family
ID: |
27039368 |
Appl.
No.: |
06/529,031 |
Filed: |
September 2, 1983 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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459471 |
Jan 20, 1983 |
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Current U.S.
Class: |
435/6.11;
435/287.2; 435/34; 435/879; 536/24.3; 536/24.32; 536/25.32 |
Current CPC
Class: |
C07K
14/255 (20130101); C12Q 1/689 (20130101); C12Q
1/6813 (20130101); Y10S 435/879 (20130101) |
Current International
Class: |
C07K
14/255 (20060101); C07K 14/195 (20060101); C12Q
1/68 (20060101); C12Q 001/68 (); C12Q 001/02 ();
C12N 015/00 (); C07H 021/00 () |
Field of
Search: |
;435/6,29,34,35,253,317,172.3,259 ;436/501,801,803,94,518,530
;536/27 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Stoleru, G. H. et al, Ann. Microbiol. (Inst. Pasteur), vol. 127,
No. 4, May-Jun., 1976, pp. 465, 477-486. .
Fitts, R. et al. Applied and Environmental Microbiology, vol. 46,
Nov. 1983, pp. 1146-1151. .
Moseley, S. L. et al. J. of Infectious Diseases, vol. 142, No. 6,
Dec. 1980, pp. 892-898. .
Nichols, B. P. et al. Proc. Nat'l Acad. Sci. USA, vol. 76, No. 10,
Oct. 1979, pp. 5244-5248. .
Cleary, J. M. et al. J. Bacteriology, vol. 150, No. 3, Jun. 1982,
pp. 1467-1471. .
Stoleru, G. H., et al. Chemical Abstracts vol. 85, No. 13, 1976
#90053b, p. 288. .
Langer, P. R. et al. Proc. Nat'l Acad. Sci., vol. 78, No. 11, Nov.
1981, pp. 6633-6637. .
Grunstein et al., (1975) Proc. Natl. Acad. Sci. USA 72 pp.
3961..
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Primary Examiner: Kepplinger; Esther M.
Assistant Examiner: Jay; Jeremy
Parent Case Text
This application is a continuation-in-part of our co-pending
application U.S. Ser. No. 459,471 filed Jan. 20, 1983 now
abandoned.
Claims
What is claimed is:
1. A method of detecting the presence of Salmonella in a
bacteria-containing sample comprising
providing a sample suspected of containing Salmonella,
lysing the bacteria in said sample to release their DNA,
denaturing said released DNA,
providing at least one DNA probe which is capable of stably
hybridizing to DNA from 80% or more of Salmonella species, and not
to DNA of any other enterobacteria, to form detectable
complexes,
contacting said DNA probe with said released DNA in said sample
under conditions which allow said probe to hydridize to Salmonella
DNA present in said sample to form hybrid DNA complexes, and
detecting said hybrid DNA complexes as an indication of the
presence in said sample of Salmonella.
2. The method of claim 1 wherein said probe is labeled.
3. The method of claim 2 wherein said label is either capable of
being detected or is capable of selectively bonding to an indicator
to form a detectable complex.
4. The method of claim 3 wherein said probe is labeled with a
radioactive isotope.
5. The method of claim 4 wherein said radioactive isotope is
.sup.32 P which has been incorporated into said probe by
nick-translation.
6. The method of claim 4 wherein said radioactive isotope is
.sup.125 I which has been incorporated into said probe by
nick-translation.
7. The method of claim 3 wherein said label is biotin and said
indicator is avidin to which is bonded a chemical entity which,
when said avidin is bonded to said biotin on said hybrid DNA
complex, is capable of being detected.
8. The method of claim 7 wherein said chemical entity is a
fluorophore which renders said hybrid DNA complexes
fluorometrically detectable.
9. The method of claim 7 wherein said chemical entity is an
electron-dense compound which renders said hybrid DNA complexes
detectable by an electron microscope.
10. The method of claim 7 wherein said chemical entity is an
antibody which renders said hybrid DNA complexes immunologically
detectable.
11. The method of claim 7 wherein said chemical entity is one of a
catalyst/substrate pair which renders said hybrid DNA complexes
enzymatically detectable.
12. The method of claim 1 wherein, prior to contacting said DNA
with said probe, said bacteria are separated out of said sample and
said DNA is immobilized on a DNA binding support.
13. The method of claim 12 wherein said support is a nitrocellulose
membrane.
14. The method of claim 1, wherein said probe is unlabeled and said
detection of hybrid DNA complexes is carried out by sandwich
hybridization.
15. The method of claim 1, employing at least 2 different said
probes
16. The method of claim 15, employing at least 3 different said
probes.
17. The method of claim 16, employing at least 4 different said
probes
18. The method of claim 17, employing at least 5 different said
probes.
19. A DNA probe consisting of a portion of a Salmonella chromosome
which is capable of stably hybridizing to DNA from 80% or more of
Salmonella species and not to other enterobacteria.
20. The DNA probe of claim 19 wherein said probe is labeled.
21. The DNA probe of claim 19 wherein said probe is capable of
stably hydridizing to 90% or more of Salmonella species.
22. The DNA probe of claim 19, said probe being the Salmonella DNA
BamHl fragment contained in plasmid YEp13 contained in E. coli,
said plasmid-containing E. coli being NRRL B-15480.
23. The DNA probe of claim 19, said probe being the Salmonella DNA
BamHl fragment contained in plasmid YEp13 contained in E. coil,
said plasmid-containing E. coli being NRRL B-15479, ATCC 39261.
24. The DNA probe of claim 19, said probe being the Salmonella DNA
BamHl fragment contained in plasmid YEp13 contained in E. coli,
said plasmid-containing E. coli being NRRL B-15472.
25. The DNA probe of claim 19, said probe being the Salmonella DNA
BamHl fragment contained in plasmid YEp13 contained in E. coli,
said plasmid-containing E. coli being NRRL B-15473.
26. The DNA probe of claim 19, said probe being the Salmonella DNA
BamHl fragment contained in plasmid YE13 contained in E. coli, said
plasmid-containing E. coli being NRRL B-15484.
Description
BACKGROUND OF THE INVENTION
This invention relates to the detection of bacteria of the genus
Salmonella (bacteria of the genus Salmonella refers herein to
bacteria classified as such in Buchanan et al., The Shorter
Bergey's Manual for Determinative Bacteriology (Williams &
Wilkins 1982); such bacteria will be referred to hereinafter simply
as "Salmonella").
The most commonly used test for the presence in food of Salmonella
involves the measurement of classical biological characteristics.
The test consumes several days.
SUMMARY OF THE INVENTION
In general, the invention features a method of detecting the
presence of Salmonella in a food sample including providing at
least one DNA probe which is capable of selectively hybridizing to
Salmonella DNA to form detectable complexes, contacting the DNA
probe with the bacteria in the food sample under conditions which
allow the probe to hybridize to Salmonella DNA present in the food
sample to form hybrid DNA complexes, and detecting the hybrid DNA
complexes as an indication of the presence of Salmonella in the
food sample. (The term "selectively hybridizing", as used herein,
refers to a DNA probe which hybridizes only to Salmonella, and not
to any other enterobacteria.)
In preferred embodiments of the invention, the probe is labeled,
e.g., with a radioactive isotope, e.g. .sup.32 P or .sup.125 I,
which is incorporated into the DNA probe, e.g. by
nick-translation.
In other preferred embodiments, the probe is labeled with biotin,
which reacts with avidin to which is bonded a chemical entity
which, when the avidin is bonded to the biotin, renders the hybrid
DNA complex capable of being detected, e.g., a fluorophore, an
electron-dense compound capable of rendering the hybrid DNA
complexes detectable by an electron microscope, an antibody capable
of rendering the hybrid DNA complexes immunologically detectable,
or one of a catalyst/substrate pair capable of rendering the hybrid
DNA complexes enzymatically detectable; prior to contacting the
bacteria with the probe, the bacteria are lysed to release their
DNA, which is then denatured and immobilized on an appropriate
DNA-binding support such as a nitrocellulose membrane; and the
method employs at least 2, and preferably at least 3, 4, or 5
different Salmonella-specific probes.
In other preferred embodiments, the probe is unlabeled and
detection is carried out by means of sandwich hybridization.
The Salmonella detection method of the invention employs one or,
preferably, more, Salmonella DNA probes, each of which is a
Salmonella DNA fragment common to all or most (preferably, greater
than 80%, most preferably greater than 90%) of the great number of
known Salmonella species, while at the same time being apparently
absent from all other enterobacteria. This finding, that there is a
family of highly Salmonella-specific fragments distributed
throughout the entire Salmonella genus, was extremely suprising,
particularly in view of the supposed common lineage of all
enterobacteria, including Salmonella.
The family of probes of the invention do not code for any protein
of which we are now aware, and also are not known to contribute to
pathogenicity. The method of the invention thus does not depend on
the ability to match a DNA probe with any phenotypic characteristic
of Salmonella; i.e., there is no need to use as a probe a DNA
fragment which is known to contribute any particular distinguishing
feature of the genus. As far as is known, the probes of the
invention are the first bacterial genus-specific probes with such
wide distribution throughout the genus.
Our discovery that there is not just one, but a number, of
Salmonella-specific probes provides the added advantage of
increased sensitivity and signal amplification; the larger the
number of different probes used, the greater the sensitivity of the
assay. This is because, when several different probes are used,
each can hybridize to a different portion of a single Salmonella
chromosome, so that the single chromosome bears multiple
labels.
The assay of the invention gives rapid, accurate results, allowing
food manufacturers to reduce food storage time prior to shipment.
In addition, the short time required to complete the test permits
laboratories to handle large numbers of samples in a short period
of time. Furthermore, the assay, depending only on the overall DNA
sequences of the bacteria rather than their biochemical properties,
can detect biochemically atypical as well as typical Salmonella
bacteria, so that false negatives are avoided. The test requires no
elaborate equipment and can be performed easily by personnel who
have not had extensive technical training.
Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments
thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
We first briefly describe the drawings.
DRAWINGS
FIG. 1 is an exploded isometric view of apparatus useful in
carrying out the invention.
FIG. 2 is a table of restriction maps of Salmonella-specific probes
of the invention.
FOOD SAMPLE PROCESSING APPARATUS
There is shown in FIG. 1 food sample processing apparatus 10; this
apparatus does not constitute a part of the present invention. The
apparatus includes cap 12, top disposable cylindrical portion 14,
and bottom cylindrical portion 16, the narrowed top portion 18 of
which snugly mates with the recessed bottom portion of cylindrical
portion 14.
Cylindrical portion 14 is fitted with large pore (5-200 micron)
filter 22 capable of allowing the passage of small,
Salmonella-sized bacteria while filtering out small food particles
and larger bacteria. Filter 22 is supported on large-pore, rigid
plastic grid 24.
Cylindrical portion 16 is fitted with nitrocellulose DNA-binding
membrane 26 supported on large-pore plastic grid 28, which
constitutes the floor of cylindrical portion 16. Cylindrical
portion 16 is sized to fit snugly into a cylindrical hole of the
manifold of a vacuum suction device (not shown).
The illustrated apparatus is employed in the method of the
invention as follows. A sample of food suspected of containing
Salmonella is placed in portion 14. Portions 14 and 16 are fit
snugly together, and vacuum is applied at the bottom end of portion
16, causing Salmonella-sized bacteria to be deposited on filter 26,
while food particles and larger bacteria remain trapped in portion
14, which is then discarded. The Salmonella detection method of the
invention is then carried out on the bacteria on filter 26, as will
be described in detail below.
DNA Probes
A library of S. typhimurium DNA is constructed using the plasmid
vector YEp13, described in Broach et al. (1979) Gene 8, 121. DNA
from S. typhimurium, strain ATCC e23566 is digested to completion
with the restriction endonuclease Bam HI to create fragments of
varying lengths, and is then ligated into the unique Bam HI site of
YEp13, using the general method described in Cohen et al. (1973)
PNAS 70, 3240. This ligation mix is then used to transform either a
Leu.sup.- Saccharomyces cereviseae (yeast) host, or a culture of E.
coli strain MC1061; E. coli transformants are selected on the basis
of ampicillin resistance. The yeast and E. coli host cells are
amplified in a 500 ml volume to yield the Salmonella DNA library.
The library of Salmonella typhimurium DNA is then screened in order
to isolate restriction enzyme digestion fragments which hybridize
selectively with Salmonella DNA but do not cross-hybridize with the
DNAs of other bacteria found in food, including other bacteria in
the family Enterobacteriaceae (herein, "enterabacteria"). Screening
is carried out with genomic S. typhimurium or E. coli DNA
nick-translated with a radioactive isotope.
The above screening procedure in yeast yields a fragment which
selectively hybridizes to Salmonella DNA and not to other bacteria,
including other enterobacteria. The E. coli clone containing
plasmid YEp13 bearing this fragment was deposited in the culture
collection of the American Type Culture Collection in Rockville,
Md. on Jan. 10, 1983 identified there as RF 305, and assigned ATCC
accession number 39261.
To verify that the above fragment is common to a variety of
Salmonella cultures, this fragment is cut out of the plasmid vector
with Bam HI, purified by agarose gel electrophoresis,
nick-translated with 32.sub.P using the nick-translation method
described in Rigby et al. (1977) J. Mol. Biol. 113, 237, and
screened, in single-stranded form, against the DNAs of a number of
field isolates of Salmonella. The fragment is found to hybridize
with all of the field isolates tested.
Additional Salmonella specific probes are obtained as described
above, screening about 200 plasmid minipreps from E. coli MC1061,
rather than yeast. In more detail, the method is as follows.
First, plasmid minipreps of MC1061 transformants are prepared, by
the method of Holmes et al. (1981) Anal. Biochem 114, 193.
Restriction enzyme digests of these minipreps are electrophoresed
through agarose gels and transferred to nitrocellulose filters, as
described in Southern (1975), J. mol. Biol. 98, 503. Genomic DNA
from strain MC1061, nick-translated with alpha-(32-P)dATP or
alpha-(32-P)dCTP, is used in hybridizations with these filters, as
described in Denhardt (1966) B.B.R.C. 23, 641. Washes after
hybridization are performed at 65 degrees C. with 0.3 M NaCl/0.03M
sodium citrate for 30 minutes. Those clones containing fragments of
Salmonella DNA which do not hybridize to the E. coli probe are
further screened against a variety of Salmonella and non-Salmonella
strains to verify the specificity of these sequences for
Salmonella.
A total of 54 cloned fragments that appear not to hybridize to the
E. coli probe are obtained from the plasmid collection examined by
minipreps. These clones are then screened against 23 Salmonella
cultures and 32 non-Salmonella cultures listed in Table 1, below.
Salmonella strains (including all arizonae) mentioned herein are
described by the conventions of The Shorter Bergey's Manual of
Determinative Bacteriology, Id, such that each serotype is given a
species name. When only the serotype and not its corresponding
species name is known, the serotype is given. With the exception of
strains serotyped by the Centers for Disease Control (CDC),
Atlanta, Ga., all strains are tested for H antigen specificity
using the Spicer-Edwards system (Difco; Spratt et al., Mol. Gen.
121:347-353).
Salmonella isolates mentioned herein are summarized by 0 antigen
groups in Table 2 below; those isolates of specific interest are
described in Table 1. Non-Salmonella strains, where noted, are
isolated from foods and identified using the Enteric-tek
identification system (Difco) with tests of growth parameters on a
variety of other bacteriological media. In some of these instances,
only the genus is indicated.
TABLE 1 ______________________________________ Bacterial and Yeast
Strains Strain Description Source
______________________________________ e23566 S. typhimurium LT2
(P22) + ATCC MC1061 E. coli XY820-4A S. cereviseae leu2-3 leu2-12
can1-11 ade2-G10 DB9000 S. typhimurium pyrF146 amtA trp130 his57
zee629::Tn5 (fels-, plasmid-) S100 Salmonella a B(0):Z29(H) S102
Salmonella a G(0):Z29(H) S104 Salmonella a Cl(0):G complex(H) a
S105 Salmonella a M(0):Z10 + encomplex(H) S106 Salmonella a F(0):en
complex(H) S108 Salmonella a Cl(0):r + complex(H) S110 Salmonella a
K(0):Z4(H) S111 Salmonella a Cl(0):Z29(H) S124 Salmonella a
E2(0):i(H) S125 Salmonella a M(0):Z10(H) S126 Salmonella a F(0):K +
1 complex(H) S127 Salmonella a O(0):Z4(H) S128 Salmonella a G(0):G
complex(H) S129 Salmonella a B(0):i(H) S130 Salmonella a E2(0):en
complex(H) S131 Salmonella a I(0):b(H) S132 Salmonella a G(0):Z +
en complex(H) SA19 S. newport a SA20 S. bredeney a SA21 S. thompson
a SA22 S. infantis a SA24 S. javiana a S103A Klebsiella b S103B
Citrobacter b S107 Enterobacter b S109A Enterobacter b S109B
Enterobacter b S115A Enterobacter b S115B Proteus vulgaris b S117A
Klebsiella b S117B Proteus vulgaris b S118A Citrobacter freundi b
S118B Proteus vulgaris b S112 Enterobacter b S113 Citrobacter b
S114 Citrobacter b S116 Citrobacter b S133 Proteus vulgaris a S134
Enterobacter cloacae a S135 Citrobacter freundii a RF875 S.
treforest c RF876 S. utrecht c RF877 S. humbar c RF878 S. uccle c
RF879 S.tranora c RF880 S. artis c RF882 S. tokai c RF883 S. basel
c RF884 S. betioky c RF885 S. luton c RF886 Salmonella c
(64:k;e,n,x,z15) RF887 Salmonella c (65:-:1,6) RF888 S. brookfield
c RF889 S. crossness c RF890 S. brookfield c RF891 Salmonella c
(66:z35:-) RF892 S. bangor c RF893 Salmonella c (66:z65:-) RF894
Salmonella c (48:z41:-) RF895 Salmonella c (44:4:-) RF896 S.
simsbury c RF897 S. rutgers c RF898 S. aesch c RF899 S. crossness c
RF900 S. anarctica c RF901 S. kunzendorf c RF943 Shigella flexneri
d RF944 Shigella (Group B) d RF945 S. flexneri d RF946 Shigella
(Group B) d RF947 Shigella (Group B) d RF948 Shigella (Group C) d
RF949 Shigella (Group D) d RF950 Shigella (Group B) d RF951
Shigella (Group B) d RF952 Shigella (Group A) d RF953 Yersinia
enterocolitica d RF954 Y. enterocolitica d RF955 Y. enterocolitica
d ______________________________________ a Silliker Laboratories b
this work c Center for Disease Control d Gary Doern
Most of the 54 clones not hybridizing to E. coli do cross-hybridize
to some of the non-Salmonella cultures. Fourteen clones, including
the ten clones illustrated in FIG. 2, are found to be highly
specific for the Salmonella cultures, with no cross-hybridization
to other isolates. These are then extensively studied by using the
cloned inserts in each as probes to survey other Salmonella
isolates.
This screening is carried out as follows. First, the fourteen
Salmonella-specific fragments are cut out of the plasmid vector
using Bam HI, electrophoresed through agarose gels, electroeluted,
and labeled with .sup.32 P via nick-translation, as described
above.
Dot blots of Salmonella and other bacterial isolates to be surveyed
using the above probes are prepared by concentrating cultures of
the bacteria in L broth approximately 20-fold and then spotting
one-microliter drops onto nitrocellulose filters. The bacteria are
lysed in situ and their DNAs denatured and fixed by placing filters
sequentially onto Whatman 3M paper saturated with 0.2M NaOH/ 0.6M
NaCl and 1M Tris (pH 8.0)/0.6M NaCl, and then immersing the filters
in absolute ethanol and allowing them to dry.
The results of the above screening procedure are given in Table 2,
below. As shown therein, clone RF321 (FIG. 2) hybridizes to every
Salmonella isolate tested.
TABLE 2
__________________________________________________________________________
DISTRIBUTION OF SALMONELLA-SPECIFIC SEQUENCES WITHIN THE GENUS
SALMONELLA Number of Species or isolates NUMBER OF ISOLATES
HYBRIDIZED WITH Serotype tested RF321 RF356 RF319 RF333 RF305-1
RF304 RF344 RF318 RF347-3 RF367
__________________________________________________________________________
Group A S. paratyphi A 5 5 5 5 5 5 1 0 0 0 1 Group B Salmonella
(4,5,12:z6,-) 1 1 1 1 1 1 0 0 1 1 0 (4,12:3,n:z15) 1 1 1 1 1 1 0 0
0 0 0 S. abortus-equi 1 1 1 1 1 1 0 0 1 1 0 S. abortus-ovis 2 2 2 2
2 1 0 0 0 0 0 S. agona 3 3 3 3 3 3 0 3 2 2 0 S. bispebjerg 1 1 1 1
1 1 0 0 1 1 0 S. brandenburg 1 1 1 1 1 1 0 0 1 1 0 S. bredeney 3 3
3 3 3 3 0 2 1 1 0 S. budapest 1 1 1 1 1 1 0 1 1 1 0 S. chester 1 1
1 1 1 1 0 0 0 0 0 S. derby 4 4 4 4 4 4 0 0 4 4 0 S. essen 1 1 1 1 1
1 0 0 0 0 0 S. heidelberg 4 4 4 4 4 4 4 0 4 4 0 S. java 3 3 3 3 3 3
0 2 3 3 0 S. paratyphi B 7 7 7 7 7 7 0 5 6 6 0 S. reading 2 2 2 2 2
2 0 0 0 0 0 S. st. paul 3 3 3 3 3 3 0 0 3 3 0 S. san-diego 1 1 1 1
1 1 0 0 0 0 0 S. schleissheim 1 1 1 1 1 1 0 0 0 0 0 S. stanley 1 1
1 1 1 1 0 1 0 0 0 S. typhimurium 13 13 13 13 13 13 13 13 13 13 9
Group C1 S. amersfoort 1 1 1 1 1 1 0 0 1 1 0 S. bareilly 2 2 2 2 2
2 0 0 2 2 2 S. braenderup 4 4 4 4 4 4 0 0 4 4 4 S. cholerae-suis 6
6 6 6 6 6 0 0 6 6 4 S. hartford 1 1 1 1 1 1 0 0 1 1 0 S. infantis 5
5 5 5 5 5 0 0 4 4 0 S. livingston 1 1 1 1 1 1 1 1 1 1 0 S. mbandaka
1 1 1 1 1 1 0 0 0 0 0 S. mikawasima 2 2 2 2 2 2 0 0 2 2 0 S.
montevideo 4 4 4 4 4 4 1 2 0 0 0 S. ohio 1 1 1 1 1 1 0 1 1 1 0 S.
oranienberg 4 4 4 4 4 4 0 0 0 0 1 S. oslo 1 1 1 1 1 1 0 0 1 1 0 S.
paratyphi C 1 1 1 1 1 1 0 0 1 1 0 S. potsdam 1 1 1 1 1 1 0 0 1 1 0
S. tennesse 2 2 2 2 2 2 0 0 0 0 0 S. thompson 6 6 6 6 6 6 0 0 5 5 0
S. thphi-suis 1 1 1 1 1 1 0 0 1 1 0 S. virchow 2 2 2 2 2 2 0 0 1 1
0 Group C2 S. blockley 4 4 4 4 4 4 0 0 4 4 1 S. bovismorbificans 1
1 1 1 1 1 0 0 1 1 0 S. dusseldorf 1 1 1 1 1 1 0 0 0 0 0 S. glostrup
1 1 1 1 1 1 0 0 0 0 0 S. haardt 2 2 2 2 2 2 0 0 1 1 0 S. kottbus 1
1 1 1 1 1 0 0 1 1 0 S. litchfield 1 1 1 1 1 1 0 0 1 1 0 S. mandaka
1 1 1 1 1 1 0 0 0 0 0 S. muenchen 4 4 4 4 4 1 0 0 4 4 0 S.
narashino 1 1 1 1 1 1 0 0 1 1 0 S. newport 6 6 6 6 6 1 0 1 6 6 0 S.
tallahassee 1 1 1 1 1 1 0 0 1 1 0 S. tulear 1 1 1 1 1 1 0 0 0 0 0
Group C3 S. kentucky 1 1 1 1 1 1 0 0 1 1 0 S. virginia 1 1 1 1 1 1
0 0 1 1 0 S. bornum 1 1 1 1 1 1 0 0 0 0 0 Group D S. berta 1 1 1 1
1 1 0 0 1 1 0 S. glegdam 1 1 1 1 1 1 0 0 1 1 0 S. dar-es-saalam 1 1
1 1 1 1 0 0 1 1 0 S. dublin 1 1 1 1 1 1 0 0 1 1 0 S. eastbourne 1 1
1 1 1 1 1 1 0 0 0 S. enteritidis 4 4 4 4 4 4 0 0 4 4 4 S.
gallinarum 1 1 1 1 1 1 0 0 1 1 0 S. javiana 3 3 3 3 3 3 1 3 2 2 0
S. moscow 1 1 1 1 1 1 0 0 1 1 1 S. napoli 1 1 1 1 1 1 0 1 1 1 0 S.
panama 4 4 4 4 4 4 0 0 0 0 0 S. pensacola 1 1 1 1 1 1 0 0 1 1 0 S.
rostock 1 1 1 1 1 1 0 0 1 1 1 S. sendai 1 1 1 1 1 1 0 0 0 0 0 S.
typhi 7 7 7 7 7 7 0 0 0 0 0 S. fresno 1 1 1 1 1 1 0 0 0 0 0 S.
gateshead 1 1 1 1 1 1 0 1 0 0 0 S. strasbourg 1 1 1 1 1 1 0 0 1 1 0
Group E Salmonella (3,10:1,6:-) 1 1 1 1 1 1 0 0 1 1 0 S. anatum 4 4
4 4 4 4 0 0 4 4 0 S. butantan 1 1 1 1 1 1 0 1 0 0 0 S. give 1 1 1 1
1 1 0 1 0 0 0 S. london 2 2 2 2 2 2 0 2 0 0 0 S. meleagridis 1 1 1
1 1 1 0 0 0 0 0 S. muenster 2 2 2 2 2 2 0 2 0 0 0 S. nyborg 1 1 1 1
1 1 0 1 0 0 0 S. orion 1 1 1 1 1 1 0 0 1 1 0 S. pullorum 1 1 1 1 1
1 0 0 1 1 0 S. arkansas 1 1 1 1 1 1 0 1 1 1 0 S. newington 1 1 1 1
1 1 0 0 1 1 0 S. illinois 2 2 2 2 2 2 0 0 2 2 0 S. menneapolis 1 1
1 1 1 1 0 0 1 1 0 S. new brunswick 3 3 3 3 3 0 3 3 3 3 0 S.
chittagong 1 1 1 1 1 1 0 0 1 1 0 S. krefeld 1 1 1 1 1 1 1 1 1 1 0
S. luciana 1 1 1 1 1 1 0 1 1 1 0 S. seftenberg 2 2 2 2 2 2 0 0 2 2
0 S. westerstede 1 1 1 1 1 1 0 0 1 1 0 Groups F,G S. aberdeen 1 1 1
1 1 1 0 0 1 1 0 S. rubislaw 2 2 2 2 2 2 0 2 2 2 0 S. marshall 1 1 1
1 1 1 0 0 0 0 0 S. poona 3 3 3 3 3 3 3 3 0 0 0 S. havana 1 1 1 1 1
1 0 0 1 1 0 S. mississippi 1 1 1 1 1 1 0 1 0 0 0 S. wichita 1 1 1 1
1 1 0 0 1 1 0 S. worthington 2 2 2 2 2 2 0 0 0 0 0 Groups H, I, J,
S. boecker 1 1 1 1 1 1 0 1 0 0 0 S. carrau 1 1 1 1 1 1 1 1 0 0 0 S.
onderstepoort 1 1 1 1 1 1 0 1 0 0 0 S. florida 1 1 1 1 1 1 1 1 0 0
0 S. horsham 1 1 1 1 1 1 0 1 0 0 0 S. sundsvall 1 1 1 1 1 1 1 1 0 0
0 S. gaminara 1 1 1 1 1 1 0 0 0 0 0 S. nottingham 1 1 1 1 1 1 0 1 0
0 0 S. kirkee 1 1 1 1 1 1 0 1 1 1 0 S. cerro 5 5 5 5 5 5 3 1 4 4 0
Groups L, M, N S. minnesota 2 2 2 2 2 2 0 2 0 0 0 Salmonella 1 1 1
1 1 1 0 0 0 0 0 (28:y:-) 1 1 1 1 1 1 0 0 0 0 0
S. babelsberg 1 1 1 1 1 1 0 0 0 0 0 S. dakar 1 1 1 1 1 1 0 1 0 0 0
S. pomona 1 1 1 1 1 1 0 0 0 0 0 S. matopeni 1 1 1 1 1 1 0 0 0 0 0
S. morehead 1 1 1 1 1 1 0 0 0 0 0 S. ramat-gan 1 1 1 1 1 1 1 1 1 1
0 S. soerenga 1 1 1 1 1 1 0 1 1 1 0 S. sternschanze 1 1 1 1 1 1 0 0
0 0 0 S. urbana 2 2 2 2 2 2 0 0 0 0 0 S. wayne 1 1 1 1 1 1 0 0 0 0
0 Groups O, P, Q S. adelaide 2 2 2 2 2 2 0 0 2 2 0 S. alachua 1 1 1
1 1 1 1 1 1 0 S. monshaui 2 2 2 2 2 2 0 2 2 2 0 S. emmastad 1 1 1 1
1 1 0 0 1 1 1 S. freetown 1 1 1 1 1 1 0 0 0 0 0 S. inverness 2 2 2
2 2 2 0 2 0 0 0 S. lansing 1 1 1 1 1 1 0 0 1 1 0 S. champaign 2 2 2
2 2 2 0 2 2 2 0 Groups R, S, T S. bern 1 1 1 1 1 1 0 0 0 0 1 S.
bulawayo 1 1 1 1 1 1 0 0 0 0 0 S. johannesburg 1 1 1 1 1 1 0 0 0 0
0 S. karamoja 1 1 1 1 1 1 0 0 1 1 0 S. riogrande 1 1 1 1 1 1 0 1 0
0 0 S. springs 1 1 1 1 1 1 0 0 0 0 0 S. waycross 1 1 1 1 1 1 0 1 1
1 0 S. weslaco 2 2 0 2 2 2 0 0 0 0 0 Groups U, V, W S. berkeley 1 1
1 1 1 1 0 0 1 1 0 S. bunnik 2 2 2 2 2 2 0 0 2 2 0 S. kingabwa 2 2 2
2 2 2 0 0 2 2 0 S. guinea 1 1 1 1 1 1 0 0 0 0 0 S. niarembe 1 1 1 1
1 1 0 1 0 0 0 S. deversoir 1 1 1 1 1 1 0 0 0 0 0 S. dugbe 1 1 1 1 1
1 0 1 0 0 0 Groups X, Y, Z S. bere 1 1 1 1 1 1 0 1 1 1 0 S. bergen
1 1 1 1 1 1 1 1 0 0 0 S. kaolack 1 1 1 1 1 1 0 0 0 0 0 S. quimbamba
1 1 1 1 1 1 1 0 0 0 S. quinhon 1 1 0 1 1 1 0 0 0 0 S. dahlem 1 1 1
1 1 0 1 0 0 0 S. djakarta 1 1 1 1 1 1 0 0 0 0 0 S. flint 1 1 1 1 1
1 0 0 0 0 0 S. greenside 1 1 1 1 1 1 0 0 0 0 0 S. hooggraven 1 1 1
1 1 0 0 0 0 0 0 S. wassenaar 2 2 2 2 2 0 0 0 0 0 0 Higher O Groups
S. treforest 1 1 1 1 1 1 0 1 0 0 0 S. utrecht 1 1 1 1 1 1 0 0 1 1 0
S. humber 1 1 1 1 1 1 0 1 0 0 0 S. uccle 1 1 0 1 1 1 0 0 1 1 0 S.
tranoroa 1 1 1 1 1 1 0 0 0 0 0 S. artis 1 1 1 1 1 1 0 1 0 0 0 S.
tokai 1 1 1 1 1 0 1 0 0 0 0 S. basel 1 1 1 1 1 1 0 0 0 0 0 S.
betioky 1 1 1 1 1 1 0 0 0 0 0 S. luton 1 1 1 1 1 1 0 0 0 0 0 S.
brookfield 2 2 2 2 0 0 0 1 0 0 0 S. crossness 1 1 1 1 1 1 0 0 1 1 0
S. bangor 1 1 1 1 0 0 0 0 0 0 0 S. simsbury 1 1 1 1 1 1 0 0 0 0 0
Salmonella (64:k:3,n,x,z15) 1 1 1 1 1 0 0 1 0 0 0 (65:-:1,6) 1 1 1
1 1 1 0 0 0 0 0 (66:z35:-) 1 1 1 1 1 0 0 0 0 0 0 (66:z65:-) 1 1 1 1
1 0 0 1 0 0 0 (48:z41:-) 1 1 1 1 1 0 0 0 0 0 0 (44:4:-) 1 1 1 0 1 0
0 1 0 0 0 S. arizona diphasic 11 11 11 11 11 11 1 3 0 0 4
monophasic 30 30 30 30 30 30 0 0 0 0 23
__________________________________________________________________________
*Number of isolates giving a positive result are listed.
Clones RF356, RF352, RF354, and RF355-1, like RF305-1, hybridize to
all Salmonella isolates (clone RF305, deposited in the ATCC, has
since been renamed RF305-1). Clones RF321, RF352, RF354, and
RF355-1 are found to contain identical inserts, although at least
three are independent clones. RF321 and RF354 contain the same Bam
HI fragment, although in opposite orientations relative to the
plasmid vector. RF355-1 is derived from a clone which contains, in
addition, a second Bam HI fragment which is 1.4 KB in size; the
orientation of the 5.6 KB fragment is the same as for RF354. RF352
is identical to RF321. Clone RF356 differs from the others by size
and restriction map.
Clones RF319, RF333, RF326, and RF305-1 hybridize to fewer than
100% of all Salmonella isolates surveyed. By size, restriction
enzyme map, and hybridization pattern, clones RF333 and RF326 are
identical, except that they have opposite orientations in the
plasmid vector.
Five of the fragments illustrated in FIG. 2 were deposited in the
Agricultural Research Culture Collection (NRRL), International
Depository Authority, Peoria, IL, on June 29, 1983. Each fragment
is contained in plasmid YEp13, contained in E. coli. The NRRL
identifying numbers of the fragments are as follows:
RF 356: NRRL B-15484;
RF 333: NRRL B-15473;
RF 319: NRRL B-15472;
RF 305: NRRL B-15479;
RF 321: NRRL B-15480.
Clones RF304, RF344, RF318, RF347-3, and RF367 hybridize to 50% or
less of all Salmonella isolates tested, and are thus much less
suitable as probes than the probes of FIG. 2, which are almost
certainly chromosomal, consisting of a portion less than the entire
chromosome. For RF344 and RF347-3, the hybridization patterns are
different from each other. RF318 has the same distribution as RF
347-3, but differs in size and restriction map. Clones RF304 and
RF367 have narrow distribution in the genus. However, the
hybridization patterns for these two clones are different. All of
these clones hybridize to DNA from DB9000, an S. typhimurium LT2
strain which lacks the cryptic plasmid typically found in S.
typhimurium. Because the distribution of these cloned fragments
varies extensively, RF304, RF344, and RF347-3 may be parts of
different phages or plasmids; if so, RF318 and RF347-3 are probably
derived from the same plasmid or bacteriophage.
Quantititive Salmonella Detection
Before describing in detail a method for using the probes of the
invention to detect Salmonella, it must be pointed out that
detection of the hybrid complexes formed betweeen the probes of the
invention and Salmonella DNA can be accomplished in a variety of
ways. One method is to label the probes, so that the hybrid
complexes will also be labeled. The probes can be labeled in any of
a variety of ways. Some labeling methods are direct, i.e., the
label which is bonded to the probe is itself detectable; examples
are radioactive isotopes. Other labels are indirect, i.e., the
label is not itself detectable until it undergoes one or more
reactions following hybridization; an example is a compound such as
biotin, a label which is not itself detectable, but becomes
detectable after it reacts with avidin bound to a detectable
chemical entity such as a fluorophore, or an enzyme such as
horseradish peroxidase (HRP). In the case of HRP, detection is
accomplished via a substrate for HRP; a preferred such substrate is
the chromogen diaminobenzidine.
For convenience, then, the term "label" as used herein refers to
directly detectable entities such as raoioactive isotopes, as well
as to indirectly detectable entities such as biotin. The entity,
e.g., HRP/avidin, to which an indirectly detectable label bonds to
become detectable is referred to herein as an "indicator".
The most preferred labels for the probes are non-isotopic labels
attached to the cytosine and adenine bases of the probes via
linking groups. Such labeling is described in Landes U.S. Ser. No.
529,044 entitled "Labeled DNA", filed on the same day as this
application, assigned to the same assignee as this application,
hereby incorporated by reference.
The method of non-isotopic labeling described in the above Landes
Pat. Appln. requires that a cross-linking reagent be used to join
an enzyme molecule to the DNA; for this reaction to be carried out,
the DNA must be in single stranded form. It is convenient, if this
type of labeling is to be used, to maintain the probe in single
stranded form, prior to labeling. This can be done by incorporating
the single stranded probe into a phage vector, e.g. the publicly
available phage vector Fd., described in Zindr et al. (1982) Gene
19, 1, using the method described therein, which involves adding
Hind III linkers to the ends of a probe and then cloning the probe
into the unique Hind III site of phage vector Fd. When the single
stranded probe is to be labeled it is cut out of Fd. using Hind III
restriction enzyme.
If the probes are to be isotopically labeled, they can conveniently
be maintained in a plasmid vector, e.g. YEp13, prior to labeling.
Labeling most conveniently involves cutting out of the plasmid the
probe to be used in the hybridization assay and then
nick-translating the probe. Alternatively, the entire plasmid can
be nick-translated and used in the assay. In either case, the
probe, which in the plasmid is double-stranded, must first be
rendered single-stranded, e.g., by heat treatment or by treatment
with a base.
Another detection method, which does not require the labeling of
the probe, is the so-called sandwich hybridization technique,
described in European Pat. Appln. No. 0079139, hereby incorporated
by reference. In this assay, an unlabeled probe, contained in a
single-stranded vector, hybridizes to Salmonella DNA, and a
labeled, single-stranded vector, not containing the probe,
hybridizes to the probe-containing vector, labeling the whole
hybrid complex.
Any of the Salmonella-specific probes of the invention, labeled
with, e.g., .sup.32 P, are used to demonstrate the presence of
Salmonella in a mixture of bacteria, as follows. A pure culture of
S. typhimurium is serially diluted and then spotted onto a series
of nitrocellulose membranes. A parallel set of membranes is spotted
with the same serial dilutions of S. typhimurium, each of which is
mixed with a constant amount of E. coli. The bacteria are lysed and
their DNA denatured, e.g. by immersion of the membranes in
NaOH/NaCl, and the denaturing solution neutralized, e.g. by a
second immersion in Tris/NaCl. The bacterial DNA is then fixed onto
the membranes by immersing the membranes in absolute ethanol and
then allowing them to dry. The membranes are then soaked at
37.degree. C. for 2 hr. in prehybridization solution, as described
in Denhardt (1966) BBRC 23, 641. The prehybridization solution
consists of 45% formamide, 25 mM Na pH 6.8, 5 X Denhardt's solution
(1 X is 0.02%, w/v, of polyvinyl pyrolidone, Ficoll 500, and bovine
serum albumin), and 250 ug/ml sonicated, denatured carrier DNA
(from, e.g., calf thymus or salmon sperm). Hybridization is then
carried out, with one or more of the DNA probes described above,
using a conventional hybridization technique such as that described
in Grunstein et al. (1975) PNAS USA 72, 3961 or Falkow et al. U.S.
Pat. No. 4,358,535, hereby incorporated by reference. The
hybridization solution has the same composition as the
prehybridization solution, above, except that it further contains
10%, w/v, dextran sulfate and 0.1-10 ug/ml labeled probe.
The hybridization reaction is allowed to proceed for 2 hrs. at
37.degree. C. Non-hybridized probe is then removed by repetitive
washes of the solid support with an established wash regimen, e.g.,
3 washes of 10 minutes each with 10 mM NaCl at 37.degree. C.
Labeled DNA complexes fixed to the membranes quantitatively
correspond to the amount of Salmonella on each membrane. For each
dilution, the same quantitative result is obtained for the pure
Salmonella samples as for those containing. E. coli, demonstrating
that the presence of E. coli DNA does not affect the hybridization
of the probe to the Salmonella DNA present, and that the Salmonella
specific probes of the invention can be used to detect the presence
of Salmonella in a mixture of bacteria.
Although good results can be obtained using only one of the probes
of the invention, we have found, as discussed above, that best
results are obtained using more than one different probe at the
same time.
Detection of Salmonella in Food
The procedure for detecting the presence of Salmonella in food can
be summarized as follows.
Fifteen 25-gram randomly selected samples of food to be tested are
cultured in nutrient broth for 20-24 hours at 30-37.degree. C.
Referring to FIG. 1, a one ml aliquot of the culture is then
pipetted into cylindrical portion 14 and a vacuum applied, as
described above, so that small Salmonella-sized bacteria are
collected on nitrocellulose filter 26; portion 14, containing food
and large bacteria, is then discarded.
Next, a solution, e.g. NaOH/NaCl, which is capable of both lysing
bacteria and denaturing bacterial DNA, is added to portion 16.
Following denaturation, Tris/NaCl is added to neutralize the
NaOH/NaCl.
The bacterial DNA is then fixed onto membrane 26 by adding absolute
ethanol to the membrane and then allowing it to dry, as described
in Groet et al. U.S. Pat. Appln. Ser. No. 448,979, filed on Dec.
13, 1982, assigned to the same assignee as this application, hereby
incorporated by reference.
Following fixation, membrane 26 is soaked for 15-30 min. in
pre-hybridization buffer, described above. Hybridization buffer,
containing .sup.32 P-labeled probe, is then added, as described
above, and hybridization is allowed to proceed for 2-3 hrs. at
37.degree. C. Radioactive hybrid DNA complexes indicate the
presence in the food sample of Salmonella.
The above procedure can be used to detect the presence of
Salmonella in any foods, including all of the foods listed in Table
2.
The processing of the food samples prior to the hybridization assay
is carried out according to the methods described by the Food and
Drug Administration in U.S. FDA, Bureau of Foods, Div. of
Microbiol. (1978) Bacterialog. Anal. Manual, 5th Ed., Wash., D.C.
Association of Anal. Chem.
As indicated in Table 2, some of the food cultures tested were
inoculated with predetermined numbers of Salmonella prior to the
overnight incubation. In those cases, Salmonella cultures were
grown to approximately 2.times.10.sup.8 cells/ml in L broth,
diluted in L broth, and added to the food cultures.
All five of the NRRL-deposited probes, nick-translated with .sup.32
P, were used to assay, using nucleic acid hybridization, the
following foods (samples of food tested were both those which had
been inoculated with Salmonella, and uncontaminated samples):
peanut butter, soy flour, macaroni, chocolate pieces, nonfat dry
milk, thawed frozen fish sticks, dried eggs, dog treats, sour
cream, and instant mashed potatoes.
Inocdated samples of each type of food give very strong
hybridization results, while uncontaminated samples give very clean
negative results.
To determine whether different Salmonella strains commonly found in
food behave similarly in hybridization assays, soy flour cultures
are inoculated with about 1,000 organisms of four of the Salmonella
strains most commonly found in food, human clinical samples, and
other animals, according to the 1980 Salmonella Surveillance Annual
Summary of the Center for Disease Control, issued December, 1982.
These four strains are S. typhimurium; S. derby; S. heidelberg, and
S. st paul. The soy cultures are incubated overnight. Replicate
filters are prepared from each culture and assayed using the five
NRRL-deposited probes, nick-translated with .sup.32 P. The filters
are exposed to film for autoradiography and later counted in a
scintillation counter.
Some strains of Salmonella (e.g., S. heidelberg) may behave
differently in the hybridization assay to the extent that the
intensity of hybridization is less than that for an equal number of
Salmonella of another strain. However, this difference does not
appear to be probe-dependent, and the difference may result from
some mechanical phenomenon such as inadequate lysis of the bacteria
on the filters, causing less DNA to be exposed during the
hybridization reaction. Differences may be diminished by using a
greater amount of probe DNA in the assays. Despite this lower
hybridization intensity, S. heidelberg can be easily detected in
this assay using any or all of the Salmonella-specific probes.
Thus diverse food types can be handled with ease in this test. An
overnight period of incubation of these foods in nutrient broth is
desirable, but selective enrichment is not required.
The advantages of this system are multifold. The time required for
the analysis of a single food sample is greatly reduced from the
5-7 days currently required for a microbiological assay. In
addition, a larger number of food samples can be processed in a
small period of time. In these studies the background hybridization
is quite low, which makes possible a clear distinction between
positive and negative results. The probes described herein can also
be applied in a clinical setting to test for the presence of
Salmonella in stools; stool samples can be processed in the same
way as food samples.
Other Embodiments
Other embodiments are within the following claims. For example, as
has been mentioned, probes can be labeled using a variety of
labels. Nitrocellulose membranes are preferred for binding DNA, but
any suitable DNA-binding support, e.g. diazobenzyloxymethyl paper,
can be used.
* * * * *